Following administration, drugs are transported in biological fluids (e.g. in blood) partly in solution as free drug and partly bound to blood components (e.g., plasma proteins, blood cells). The physiologically active substances are in equilibrium between a free form and a form bound to endogenous ligands present in the same fluids (see reviews by Kremer, et al. Pharmacol Rev. 1988, 40:1–47). Only free drug is available for passive diffusion to target tissue sites where the desired biological activity may take place. When compared to the total-substance level, the free drug concentration is more closely related to drug concentration at the active site, to drug effects, and to clinical effectiveness. Observations made on both healthy and pathologically afflicted humans confirmed that the patients' clinical condition correlates better with variations in free form concentration when compared to variations in total substance concentrations.
Slight changes in the binding affinity of drugs to blood components can result in significant changes in clinical response or can even cause a toxic response. Since it is the free drug in plasma which equilibrates with the pharmacologically active site, a slight change in the binding affinity, such as from 99 to 98% binding, can result in an almost 100% change in free drug concentration, and, thus, can cause a significant alteration in response. This is the case for most HIV protease inhibitors (PIs) (Acosta, Acquir Immune Defic Syndr. 2002 Feb. 1; 29 Suppl 1:S11–8; Sadler, et al. Antimicrob Agents Chemother. 2001 March; 45(3):852–6; Anderson, et al, AIDS. 2000 Oct. 20;14(15):2293–7; Bilello, et al., J Infect Dis. 1995 March;171(3):546–51).
The binding of drugs to plasma proteins may influence their distribution, elimination and pharmacological effect, which is considered more closely related to unbound rather than total drug concentration (du Souich et al, 1993, Clin Pharmacokinet. 24:435–40.). Human serum albumin (HSA) and human α1-acid glycoprotein (AAG) are two mainly involved proteins in the binding of HIV PIs in plasma. Human AAG is an acute-phase protein whose expression increases during acute inflammatory episodes, infections, injuries, neoplastic disease, and AIDS (Kremer et al, Pharmacol Rev. 1988, 40: 1–47; Oie et al, 1993, J Acquir Immune Defic Syndr Hum Retrovirol. 5:531–533; Mackiewicz et al, 1995, Glycoconj. J. 12:241–247; van Dijk et al, 1995, Glycoconj J. 12:227–233). The level of AAG in human serum fluctuates between 0.15 and 1.5 mg/mL, and the average value may vary by as much as 4-fold between healthy volunteers and AIDS patients (Kremer et al, Pharmacol Rev. 1988, 40: 1–47; Oie et al, 1993, J Acquir Immune Defic Syndr Hum Retrovirol. 5:531–533). Additionally, AAG concentrations have been suggested to vary by race or ethnicity (Johnson et al, 1997, J. Pharm. Sci. 86: 1328–1333). It has been reported that AAG exists as a mixture of two or three genetic variants (the A variant and the F1 and/or S variants) in the plasma of most individuals (Hervé et al, 1998, Mol. Pharmacol. 54:129–138), which present different drug binding specificities.
The question whether AAG binding had an effect on in vivo antiviral activity of anti-HIV PIs has recently heightened the great interest in the investigations of effects of serum proteins on activity and pharmacokinetics of anti-HIV PIs in vitro (Billello et al. 1995, J. Infect. Dis. 171:546–551; Bilello et al. 1996, Antimicrobiol. Agents Chemother. 40:1491–1497; Lazdins et al. 1996, J. Infect. Dis. 175:1063–1070; Kiriyama et al. 1996, Biopharmac. Drug Dispos. 17:739–751; Zhang et al. 1999, J. Infect. Dis. 180:1833–1837; Jones et al. 2001, Br J Clin Pharmacol. 51:99–102; Kageyama et al. 1994, Antimicrob Agents Chemother. 22:499–506; Livingstone et al. 1995, J. Infect Dis. 172:1238–45), and in vivo (Sadler et al. 2001, Antimicrob. Agents Chemother. 45:852–856.). In vitro these studies have consistently demonstrated that human AAG reduced the antiviral activity of most PIs by decreasing the amount of free drug available for interaction with the drug target. Studies in vitro by Bilello et al. (1995, J. Infect. Dis. 171:546–551; 1996, Antimicrobiol. Agents Chemother. 40:1491–1497) have shown that the antiviral efficacy of two HIV PIs, A77003 and A80978, decreased as the concentration of AAG was increased and that the inhibition of HIV protease was highly correlated with the amount of intracellular inhibitor. Also, the clinical significance of these effects in vitro was shown by the lack of clinical efficacy of the HIV PI SC-52151, which has potent antiretroviral activity in vitro but inactivity in vivo, because extensive protein binding prevented intracellular diffusion (Fischl et al. J Acquir Immune Defic Syndr Hum Retrovirol 1997, 15:28–34). While there are extensive data to address this problem, no general correlation between protein binding and anti-HIV activity can be made so far on the basis of these studies.
Although the precise site of action of PIs has not been defined, the inhibition of HIV protease possibly takes place intracellularly. Bilello et al. (Bilello et al, 1996, Antimicrobiol. Agents Chemother. 40:1491–1497) have demonstrated that cellular uptake of protease inhibitor is proportionally decreased in the presence of AAG, which results in a decreased antiviral activity. These observations indicate that not only the antiviral EC50 (50% effective concentration) of PIs, but also their interaction with human AAG, probably are the most important determinant factors of their anti-HIV efficacy in vivo because only free drug in plasma can equilibrate with intracellular compartments.
In determining therapeutic amounts and subsequent dosage regimens for individual protease inhibitors, it is clinically relevant to establish the binding affinities of the different protease inhibitors to plasma proteins. Also of interest is knowledge about plasma protein binding sites and the free drug concentrations. These factors and the information extracted therefrom can aid in obtaining a more complete pharmacokinetic profiling of protease inhibitors, which can result in more accurate and effective therapeutic amounts and dosage regimens for the protease inhibitors, which can ultimately translate into an improved treatment for HIV infected patients.
To date, binding of a particular protease inhibitor to plasma proteins is expressed as a percentage of total amount of drug that is bound to the plasma proteins (Sadler, et al. Antimicrob Agents Chemother. 2001 March;45(3):852–6; Anderson, et al, AIDS. 2000 Oct. 20;14(15):2293–7; Bilello, et al., J Infect Dis. 1995 March;171(3):546–51). This is number for a particular concentration of drug and plasma protein. It is well established that the concentration of plasma proteins, in particular the two most important ones in HIV/AIDS therapy AAG and serum albumin (HSA), is variable depending on age, race, and disease state (Kremer, et al. Pharmacol Rev. 1988 March;40(1):1–47). Furthermore, total drug concentration is also different in individuals due to the their various rate of absorption, distribution, metabolism, and excretion. Therefore, the use of percentage of bound and free forms is not useful and cannot be generally applied. However, if the dissociation constant, Kd, is directly determined for the binding of drug to the plasma protein, and the total drug and plasma concentration is known from pharmacokinetic studies, the concentrations of the free and bound form of drug can be readily calculated (Wyman, J. and Gill, S., “Binding and Linkage”, 1990, Published by University Science Books, Mill Valley, Calif.).
Although considerable literature regarding the effect of AAG-binding on antiviral PIs has arisen (Billello et al. 1995, J. Infect. Dis. 171:546–551; Bilello et al. 1996, Antimicrobiol. Agents Chemother. 40:1491–1497; Lazdins et al. 1996, J. Infect. Dis. 175:1063–1070; Kiriyama et al. 1996, Biopharmac. Drug Dispos. 17:739–751; Zhang et al. 1999, J. Infect. Dis. 180:1833–1837; Jones et al. 2001, Br J Clin Pharmacol. 51:99–102; Kageyama et al. 1994, Antimicrob Agents Chemother. 22:499–506; Livingstone et al. 1995, J. Infect Dis. 172:1238–45), most methods for addressing this problem mainly use equilibrium dialysis, ultracentrifugation and ultrafiltration. To our knowledge, the binding affinities of PIs with AAG have not been determined using calorimetric methods. Moreover, the measurement of the thermodynamic parameters of a binding process provide a more realistic model.
In view of the clinical significance and the medical need to pharmacokinetically characterize protease inhibitors, a convenient and reliable method to measure the equilibrium dissociation constant, or a functional equivalent thereof, for the binding of a particular drug and plasma protein was designed. With the knowledge of the equilibrium dissociation constant, the in vivo activity of a particular PI in the presence of plasma proteins can be estimated using the EC50s from in vitro assays without plasma proteins. This will allow as well for an improved preclinical evaluation and selection of new PIs for future clinical development. Thus, the present invention concerns a method for determining the binding affinity of protease inhibitors to plasma proteins that is direct, has high sensitivity, and is easy to perform using routine laboratory procedures.
The present invention provides a method that can quantitatively calculate free drug concentrations of protease inhibitors in human plasma, as well as a method to calculate therapeutic amounts and dosage regimens.
Furthermore, the present invention provides a method that can calculate the effect of plasma proteins on the antiviral activity (EC50 values) of protease inhibitors from their binding affinities to plasma proteins. The present invention provides as well a method that can evaluate the in vivo anti-HIV efficacy of PIs in human plasma.
Often there may be competition between drugs in plasma protein binding, in which agents that are bound tightly, such as coumarin anticoagulants, macrolide or lincosamide antibiotics that bind tightly to alpha-1-acid Glycoprotein (AAG), are able to displace less tightly bound compounds from their binding sites and thus can increase the free form of the drug and improve the biological efficacy (Sommadossi, et al., 1998 U.S. Pat. No. 5,750,493). Therefore, the present invention provides as well a method for selecting compounds that competitively bind with plasma proteins, said selection being useful for co-administering agents to compete for plasma protein binding, so that an increase of the free plasma concentration of protease inhibitors can be achieved.